Abstract Mechano-activation of both reactive oxygen species (ROS) and calcium (Ca2+) signaling has been widely reported in diverse cell types, including skeletal muscle. We are now investigating the mechanisms by which mechano-activated ROS and Ca2+ influence the physiology of normal muscle, as well as the pathophysiology following their dysregulation in muscular dystrophy. This proposal builds upon our recent provocative discovery and characterization of X-ROS signaling which suggested that many critical elements of mechano-signaling arise from the microtubules, their mechano-activation of NADPH oxidase type 2 (NoX2) ROS production (i.e., X-ROS), and its subsequent activation of sarcolemmal Ca2+ channels. We demonstrated that acutely targeting X-ROS components provided a therapeutic benefit in pre-clinical models of muscular dystrophy. Based on our previous research, we have devised a set of specific aims, using both control and genetically altered mice, which will extend our previous discoveries and further characterize the physiologic relevance of X-ROS signaling. The proposed single cell studies are enabled by state-of-the-art techniques, including many newly devised within the laboratory, that allow simultaneous measurements of force, intracellular [Ca2+], Ca2+ influx, and [ROS]i while controlling sarcomere length. These, combined with advanced biochemistry and immunofluorescence, allow the advancement of relevant findings to in vitro whole muscle and in vivo animal studies designed to inform future pre-clinical efficacy studies.